Not applicable.
The invention relates to plant enzymatic activity, and aspects thereof, involved in the biosynthesis of Coenzyme A. The invention particularly relates to the plant enzyme known either as pantoate-xcex2-alanine ligase (EC 6.3.2.1), pantoate activating enzyme or pantothenate synthetase (PS). PS catalyses the synthesis of pantothenate.
PS is an essential enzyme in the in planta biosynthesis of the vitamin and Coenzyme A precursor pantothenate. It is known to catalyse the following reaction:
ATP+(R)-pantoate+xcex2-alaninexe2x86x92AMP+pyrophosphate+(R)-pantothenate.
PS genes have previously been isolated from Escherichia coil (GenBank accession number P31663), Bacillus subtilis (GenBank accession number P52998), and the cyanobacterium Synechocystis (GenBank accession number U44896). DNA sequences from Saccharomyces cerevisiae (GenBank accession number P40459) and Schizosaccharomyces pombe (GenBank accession number Q09673) having unknown functions have been proposed to code for PS enzymes based on DNA and deduced amino acid sequence similarities. To date, however, no gene has been reported which codes for the PS enzyme in any plant species. It is therefore an object of the invention to identify, isolate and sequence a gene coding for the PS enzyme present in plants.
A number of assays have been reported for measuring PS activity. One assay developed by Maas (1950a and 1950b) uses a microbiological assay of pantothenate based on the ability to promote growth of an E. coli pantothenate auxotroph (M99-1, panC). The assay developed by Pfleiderer et al (1960) measures the AMP liberated in the PS reaction. In this assay, myokinase catalyses the production of 2 moles of ADP for each mole of AMP released in pantothenate synthesis using ATP supplied in the assay mixture. Pyruvate kinase then generates 2 moles of pyruvate and ATP for 2 moles of phosphoenolpyruvate and ADP. Finally, lactate dehydrogenase reduces 2 moles of pyruvate to yield 2 moles of lactate concomitant with stoichiometric oxidation of NADH to NAD, which can be monitored spectrophotometrically by following the absorbance at 340 nm. A third assay, developed by Miyatake et al (1979), employs an assay mix containing 14C-xcex2-alanine and unlabelled pantoate. In this assay any 14C-pantothenate formed is separated from unreacted 14C-xcex2-alanine by cation exchange chromatography and subsequently quantified by liquid scintillation counting. These assays, however, are not suitable for use with high throughput biochemical screening and cannot be used for the large scale biochemical screening of compounds necessary to discover useful inhibitors of PS.
We have developed an invention which addresses the above-mentioned drawbacks associated with the prior art. Our invention covers a number of related aspects which encompass the same inventive concept.
According to a first aspect of the invention there is provided an isolated DNA molecule encoding a protein from a plant, which protein has PS activity. In preferred embodiments, the DNA is isolated from Lotus japonicus or Oryza sativa. 
To support our invention we herein disclose the cDNA sequence from Lotus japonicus. In addition, we have shown that a previously unassigned expressed sequence tag of Oryza sativa (GenBank accession number D25017) is part of a cDNA coding sequence for a PS enzyme in Oryza sativa and disclose, as part of this invention, the full cDNA sequence of the PS gene from Oryza sativa. Furthermore, we have confirmed by sequence similarity, functional complementation of an Escherichia coli mutant devoid of PS enzyme activity, and by enzyme assays that the DNA sequence from Saccharomyces cerevisiae (GenBank accession number P40459) putatively ascribed as coding for a PS enzyme does code for the PS enzyme of S. cerevisiae. A cDNA sequence coding for a PS enzyme in L. japonicus is provided in FIG. 2 (SEQ. ID NO:1). A cDNA sequence coding for a PS enzyme in O. sativa is provided in FIG. 4 (SEQ ID NO:2). A DNA sequence coding for a PS enzyme in S. cerevisiae is provided in FIG. 8 (SEQ ID NO:3). As a result of our invention it is now possible to obtain the DNA coding sequence for the PS enzyme(s) from any plant source using methods available to those skilled in the art.
A further preferred embodiment of this aspect of our invention is an isolated DNA molecule encoding a protein from L. japonicus having PS activity wherein said protein comprises the amino acid sequence set forth in FIG. 2 (SEQ ID NO:4). A still further embodiment is an isolated DNA molecule encoding a protein from O. sativa having PS activity wherein said protein comprises the amino acid sequence in FIG. 4 (SEQ ID NO:5).
In addition, we have extended our invention to include a further aspect so as to provide a non-naturally occurring chimeric gene comprising a promoter operably linked to a DNA molecule encoding a protein from a plant having PS activity. Preferably, the protein is isolated from a dicotyledonous or a monocotyledonous plant, such as L. japonicus or O. sativa. Preferably the amino acid sequence is selected from the group set forth in FIG. 2 (L. japonicus) and FIG. 4 (O. sativa).
We have developed our invention into another aspect which provides a recombinant vector comprising a chimeric gene, wherein the vector is capable of being stably transformed into a host cell. Also comprised in this aspect is the host cell stably transformed with the vector wherein the host cell is preferably a cell selected from the group consisting of a bacterial cell, a yeast cell, and an insect cell and is further capable of expressing the DNA molecule according to the invention.
In a still further aspect we have applied our invention to the recombinant production of the PS enzyme. In particular, the invention provides a method of producing a protein having PS activity in a host organism by firstly inserting a DNA sequence encoding a protein having PS activity into an expression cassette designed for the chosen host; inserting the resultant molecule, containing the individual elements linked in proper reading frame, into a vector capable of being transformed into the host cell; growing the thus transformed host cell in a suitable culture medium; and isolating the protein product either from the transformed cell or the culture medium, or both, and purifying it.
In addition, we have developed our invention to provide methods for assaying a protein having pantothenate synthetase activity comprising; incubating pantothenate synthetase in a suitable reaction mixture in which pantothenate synthetase is capable of catalysing the conversion of pantoate, xe2x96xa1-alanine and ATP to pantothenate, AMP and pyrophosphate; determining the amount of pyrophosphate formed by a calorimetric technique based on the assay for pyrophosphate developed by Chang et al. (1983); or converting the pyrophosphate formed by the catalytic activity of the pantothenate synthetase into inorganic phosphate by the catalytic activity of an inorganic pyrophosphatase, preferably yeast inorganic pyrophosphatase; and determining the amount of inorganic phosphate generated by the catalytic activity of said inorganic pyrophosphatase by calorimetric techniques, preferably by techniques based either on the assay for inorganic phosphate developed by Lanzetta et al. (1979) or on the assay for inorganic phosphate developed by Chifflet et al. (1988).
The production of PS, for example by using the recombinant methodology described hereinabove, has enabled us to develop methods of using purified PS to screen for novel inhibitors of PS activity which may be used as herbicides to control undesirable vegetation in fields where crops are grown, particularly agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton sugar cane, sugar beet, oilseed rape, and soybeans.